Pallab Pahari

Phthalides and Phthalans: Synthetic Methodologies and Their Applications in the Total Synthesis

1. Phthalides A 1.

Baeyer-Villiger C-C bond cleavage reaction in gilvocarcin and jadomycin biosynthesis.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.

Total Synthesis of Psoralidin, an Anticancer Natural Product

A base-catalyzed condensation of phenyl acetate with acid chloride, followed by intramolecular cyclization and microwave-assisted cross-metathesis reaction, leads to the first total synthesis of psoralidin, a natural product with a broad range of biological activities, in a highly convergent and regioselective manner.

Enzymatic Total Synthesis of Defucogilvocarcin M and Its Implications for Gilvocarcin Biosynthesis

Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Go 3592 and various other Streptomyces species. GV is usually produced along with its minor congeners, gilvocarcin M (3) and gilvocarcin E (5), that vary with respect to their side chain at the C8-position.[1] Several analogues of GV (for example, 6–8, Scheme 1), which are collectively called the gilvocarcin group of natural products, have been isolated from different Streptomyces species. All of these analogues contain the characteristic, polyketide-derived benzo[d]naphtho[1,2-b]pyran-6-one chromophore, but different C-glycosidically linked sugar units Scheme 1).[2] Members of this group of natural products are well-known for strong antitumor activities,[3] a unique mode of action,[4] and remarkably low toxicities.[2c, 5] However, the inherent poor solubility of these molecules appears to be a major obstacle in their development as therapeutics. The chemical syntheses that have been developed so far are unsuitable for generating a library of analogues,[6] whereas combinatorial biosynthetic efforts have shown more promise.[7] The continued advancement and successful implementation of such combinatorial biosynthetic and mutasynthetic approaches requires an in-depth knowledge of the biosynthetic pathway. Incorporation studies with isotope-labeled precursors[8] and genetic experiments[1a, 8d, 9] have revealed that the benzo[d]naphtho[1,2-b]pyran-6-one chromophore of the gilvocarcins is produced from a polyketide-derived angucyclinone intermediate through a complex oxidative rearrangement process. However, the details of the exact sequence of biosynthetic events and the enzymes that are involved have remained elusive. In this context, we herein report a complete, one-pot, enzymatic total synthesis of defucogilvocarcin M(1), a model compound that contains the unique chromophore common to all members of the gilvocarcin group of natural products.[10] The reconstitution of this pathway then enabled further investigation into the details of the oxidative rearrangement process of GV biosynthesis by systematic variation of the enzyme mixture used. For this approach we suggest the term “combinatorial biosynthetic enzymology”.

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5′-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O2, stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be Kmα-KG = 7.5 μm, KmUMP = 14 μm, and kcat ≈ 80 min−1. The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

GilR, an Unusual Lactone-Forming Enzyme Involved in Gilvocarcin Biosynthesis

Last at last: The terminal step of the gilvocarcin V (GV) biosynthetic pathway is an unusual lactone formation. Here we show that the enzyme, GilR, dehydrogenates the hemiacetal moiety of pregilvocarcin V to the lactone found in GV by using covalently bound FAD.

Psoralidin, an Herbal Molecule, Inhibits Phosphatidylinositol 3-Kinase–Mediated Akt Signaling in Androgen-Independent Prostate Cancer Cells

The protein kinase Akt plays an important role in cell proliferation and survival in many cancers, including prostate cancer. Due to its kinase activity, it serves as a molecular conduit for inhibiting apoptosis and promoting angiogenesis in most cell types. In most of the prostate tumors, Akt signaling is constitutively activated due to the deletion or mutation of the tumor suppressor PTEN, which negatively regulates phosphatidylinositol 3-kinase through lipid phosphatase activity. Recently, we identified a natural compound, psoralidin, which inhibits Akt phosphorylation, and its consequent activation in androgen-independent prostate cancer (AIPC) cells. Furthermore, ectopic expression of Akt renders AIPC cells resistant to chemotherapy; however, psoralidin overcomes Akt-mediated resistance and induces apoptosis in AIPC cells. While dissecting the molecular events, both upstream and downstream of Akt, we found that psoralidin inhibits phosphatidylinositol 3-kinase activation and transcriptionally represses the activation of nuclear factor-κB and its target genes (Bcl-2, Survivin, and Bcl-xL, etc.), which results in the inhibition of cell viability and induction of apoptosis in PC-3 and DU-145 cells. Interestingly, psoralidin selectively targets cancer cells without causing any toxicity to normal prostate epithelial cells. In vivo xenograft assays substantiate these in vitro findings and show that psoralidin inhibits prostate tumor growth in nude mice. Our findings are of therapeutic significance in the management of prostate cancer patients with advanced or metastatic disease, as they provide new directions for the development of a phytochemical-based platform for prevention and treatment strategies for AIPC.

Enzymatic Total Synthesis of Rabelomycin, an Angucycline Group Antibiotic

A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, starting from acetyl-CoA and malonyl-CoA, using a mixture of polyketide synthase (PKS) enzymes of the gilvocarcin, ravidomycin, and jadomycin biosynthetic pathways. The in vitro results were compared to in vivo catalysis using analogous sets of enzymes.

The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.

Activating Stress-Activated Protein Kinase–Mediated Cell Death and Inhibiting Epidermal Growth Factor Receptor Signaling: A Promising Therapeutic Strategy for Prostate Cancer

Epidermal growth factor receptor (EGFR) activation is an important event that regulates mitogenic signaling, such as the Raf, mitogen-activated protein kinase (MAPK), and extracellular signal–regulated kinase 1/2 cascades. EGFR activation has been implicated in the transition of prostate cancer from androgen dependence to independence. Therefore, inhibition of EGFR may effectively suppress prostate cancer growth and progression. The goal of this study was to determine whether the natural compound psoralidin alters EGFR-mediated signaling resulting in the inhibition of prostate cancer growth. Results suggest that inhibition of EGFR alone (by serum deprivation) fails to induce stress-mediated protein kinases (SAPK), namely, Jun NH2-terminal kinase/c-Jun signaling, in androgen-independent prostate cancer (AIPC) cells. Treatment with psoralidin, however, inhibited both constitutive and EGF-induced EGFR activation and simultaneously triggered SAPK signaling, resulting in the induction of apoptosis in AIPC cells. In addition, psoralidin downregulated EGFR-regulated MAPK signaling and inhibited cell proliferation in AIPC cells. Oral administration of psoralidin effectively suppressed PC-3 xenograft tumors in nude mice. Compared with control tumors, inhibition of pEGFR expression and an increase in the phosphorylation, activation, and nuclear translocation of c-Jun were observed in psoralidin-treated tumor sections. Our studies suggest that psoralidin may be a potent therapeutic agent that modulates EGFR-mediated key epigenetic events in AIPC. Mol Cancer Ther; 9(9); 2488–96. ©2010 AACR.